Rat liver 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) has been purified to homogeneity by a rapid procedure which employs HMG-CoA affinity chromatography. Antibodies prepared against purified HMG-CoA reductase gave a single immunoprecipitin line with HMG-CoA reductase. The in vitro phosphorylation of HMG-CoA reductase was studied utilizing purified enzyme and reductase kinase. With this system phosphorylated reductase contained approximately 4 mol phosphate per tetramer of 200,000. To demonstrate that HMG-CoA reductase undergoes phosphorylation in vivo, rats were injected with 32P and hepatic reductase isolated by immunoprecipitation and by purification of the enzyme to homogeneity. Analysis of 32P labeled immunoprecipitates and purified HMG-CoA reductase by SDS gel electrophoresis revealed a single peak of radioactivity comigrating with purified reductase establishing that reductase can undergo phosphorylation in vivo. Glucagon administration in vivo resulted in a 10 fold increase in hepatic cyclic AMP content, a two-fold increase in 32P incorporation into HMG-CoA reductase, and a 40% decrease in HMG-CoA reductase enzymic activity.